High pulse wave velocity is associated with enlarged perivascular spaces in dementia with Lewy bodies

Previous studies have demonstrated associations between enlarged perivascular spaces (EPVS) and dementias such as Alzheimer’s disease. However, an association between EPVS and dementia with Lewy bodies (DLB) has not yet been clarified. We performed a cross-sectional analysis of our prospective study cohort of 109 participants (16 with DLB). We assessed cognitive function, pulse wave velocity (PWV), and brain magnetic resonance imaging features. The relationships between EPVS and DLB were evaluated using multivariable logistic regression analyses. Compared with the non-dementia group, the DLB group was more likely to have EPVS in the basal ganglia. Compared with participants without EPVS, those with EPVS were older and had cognitive impairment and high PWV. In multivariable analyses, EPVS in the basal ganglia was independently associated with DLB. High PWV was also independently associated with EPVS in both the basal ganglia and centrum semiovale. High PWV may cause cerebrovascular pulsatility, leading to accelerated EPVS in DLB participants.

www.nature.com/scientificreports/assessment of DLB from the perspective of EPVS may contribute to a better understanding of the pathophysiology of this disease.
We are currently conducting an observational study regarding cognitive function and the gut microbiota.In previous studies, we have revealed that gut microbial dysregulation is associated with cognitive decline [10][11][12] and cerebral SVD 13,14 .We are also planning to evaluate the association between gut microbiota and DLB because recent studies have assessed such a relationship 15,16 .In the present study, however, we aimed to evaluate the relationship between DLB and cerebral SVD (in particular, EPVS).We believe that an examination of cerebral SVD and the links between cerebral SVD, gut microbiota, and DLB may clarify as-yet-unidentified associations between gut microbiota and DLB.
In the present study, we thus evaluated the relationships between EPVS and DLB by performing a sub-analysis of the data from our ongoing clinical study.We hypothesized that EPVS would be associated with DLB, similar to the previously reported association between EPVS and AD.

Multivariable analyses
Multivariable logistic regression analyses revealed that BG-PVS was independently associated with the presence of DLB after adjusting for age, sex, PWV, risk factors, and the presence of cerebral SVD (odds ratio [OR], 95% confidence interval [CI] 4.29, 1.36-13.6,p = 0.012, Table 4); CS-PVS was not significantly associated with the presence of DLB.Furthermore, multivariable logistic regression analyses revealed that high PWV (PWV ≥ 19 m/s, which was the median value of the enrolled participants) was independently associated with the presence of both BG-PVS (OR 95% CI 3.84, 1.36-13.6,p = 0.031, Table 5) and CS-PVS (OR 95% CI 7.08, 2.32-21.6,p < 0.001, Table 6).

BG-PVS in DLB
Of the participants with DLB, participants with BG-PVS were more likely than those without BG-PVS to have greater cognitive impairment, a history of fall, higher Movement Disorder Society-Unified Parkinson's Disease Rating Scale (UPDRS) scores, higher PWV, and higher concentrations of plasma NfL; however, age was nearly equal between the two groups (Table S4).

Discussion
The main finding of the present study was that BG-PVS was independently associated with the presence of DLB.Furthermore, the prevalence of EPVS-both BG-PVS and CS-PVS-gradually increased with increasing cognitive impairment.We also found that higher PWV was independently associated with the presence of EPVS (both BG-PVS and CS-PVS).These findings may help to reveal the potential associations and related mechanisms between EPVS and dementia-in particular DLB-and suggest the important role of PWV in these associations.This is the first report to demonstrate associations between EPVS and PWV (more specifically, brachial-ankle PWV) in participants with DLB.Although several studies 17,18 have reported associations between aortic PWV and EPVS, these studies did not include participants with dementia.Thus, this novel link between EPVS and PWV in DLB participants provides insights into the natural history of dementia, and in particular DLB.We have www.nature.com/scientificreports/previously reported that brachial-ankle PWV, an indicator of arterial stiffness, is associated with silent lacunar infarct (SLI) 19 , white matter hyperintensity (WMH) 20 , and progressive acute lacunar infarcts 21 ; it can also predict future stroke following acute lacunar infarcts 22 .However, the ankle-brachial index was not associated with EPVS in the present study.This finding is reasonable because the ankle-brachial index indicates large-artery atherosclerosis 23 , which may be different from microvascular dysfunction.Our present data are therefore in line with the findings of previous studies and fill a knowledge gap regarding cerebral SVD-in particular EPVS-and cerebrovascular pulsatility.Recently, EPVS have been considered a component of the glymphatic system 2,6 .The glymphatic system is an emerging hypothetical system that connects perivascular spaces, allowing the exchange of waste products between the cerebrospinal fluid surrounding the brain and the interstitial fluid within the brain parenchyma 2 .The glymphatic system plays a vital role in the clearance of several metabolic products, such as amyloid-β and tau 2 .When speculating on glymphatic dysfunction, cerebrovascular pulsatility-the driving force for cerebrospinal Table 2. Comparison of background information between participants with high and low BG-PVS scores.Data are presented as medians, interquartile ranges or number of patients (%).The Wilcoxon rank-sum test and χ 2 test were used.Note that participants with high BG-PVS were defined as presenting with enlarged perivascular spaces in the basal ganglia (scores ≥ 2 based on an MRI scan at the level of the basal ganglia).ADAS-cog, Alzheimer's Disease Assessment Scale-Cognitive Subscale; APOE, apolipoprotein E; BG-PVS, enlarged perivascular spaces in the basal ganglia; BNP, brain natriuretic peptide; BP, blood pressure; CDR-SB, Clinical Dementia Rating-Sum of Boxes; CMB, cerebral microbleed; CS-PVS, enlarged perivascular spaces in the centrum semiovale; DBDS, Dementia Behavior Disturbance Scale; FAB, Frontal Assessment Battery; IADL, instrumental activities of daily living; LM-WMSR, Logical Memory subtests I and II of the Wechsler Memory Scale-Revised; MMSE, Mini-Mental State Examination; MRI, magnetic resonance imaging; NfL, neurofilament light chain; RCPM, Raven's Coloured Progressive Matrices; SLI, silent lacunar infarct; VSRAD, voxel-based specific regional analysis system for Alzheimer's disease; WMH, white matter hyperintensity.www.nature.com/scientificreports/fluid flow within the perivascular spaces-has been advocated 24 .Our finding of an association between PWV and EPVS supports the relationship between the glymphatic system and cerebrovascular pulsatility because high PWV increases cerebral microvascular pulsation 25 .Cerebrovascular pulsatility may also play an important role in hypertensive cerebral SVD.Pathological findings of WMH have been described as wall-thickened arteries, large perivascular spaces, and extensive arterial pulsations 26 .Cerebrovascular pulsatility (as detected by transcranial Doppler ultrasound) is reportedly associated with WMH 27 .We have previously proposed a "tsunami effect in the brain" as a potential mechanism of PWV in cerebrovascular pulsatility 25 .Thus, the presence of EPVS suggests overflowing compartments surrounding cerebral blood vessels and dysfunctional perivascular clearance, followed by a tsunami effect in the brain microvascular system.
In the present study, we found that BG-PVS were more common in participants with DLB.Furthermore, DLB participants with BG-PVS had more impaired motor and cognitive function and higher UPDRS scores.Motor Table 3.Comparison of background information between participants with high and low CS-PVS scores.Data are presented as medians, interquartile ranges or number of patients (%).The Wilcoxon rank-sum test and χ 2 test were used.Note that participants with high CS-PVS were defined as presenting with enlarged perivascular spaces in the centrum semiovale (scores ≥ 3 based on an MRI scan at the level of the centrum semiovale).ADAS-cog, Alzheimer's Disease Assessment Scale-Cognitive Subscale; APOE, apolipoprotein E; BG-PVS, enlarged perivascular spaces in the basal ganglia; BNP, brain natriuretic peptide; CDR-SB, Clinical Dementia Rating-Sum of Boxes; CMB, cerebral microbleed; CS-PVS, enlarged perivascular spaces in the centrum semiovale; DBDS, Dementia Behavior Disturbance Scale; DLB, dementia with Lewy bodies; FAB, Frontal Assessment Battery; IADL, instrumental activities of daily living; LM-WMSR, Logical Memory subtests I and II of the Wechsler Memory Scale-Revised; MMSE, Mini-Mental State Examination; MRI, magnetic resonance imaging; NfL, neurofilament light chain; RCPM, Raven's Coloured Progressive Matrices; SLI, silent lacunar infarct; VSRAD, voxel-based specific regional analysis system for Alzheimer's disease; WMH, white matter hyperintensity.A previous study reported that BG-PVS is associated with cognitive impairment and Parkinson's disease but that CS-PVS is not associated with cognitive impairment in Parkinson's disease participants 7 .Furthermore, higher PWV is reportedly associated with more severe dementia in DLB participants 28 .These findings are in line with the present data.Our findings are also reasonable because DLB tends to complicate autonomic dysfunction 8 , leading to blood pressure variability.Blood pressure variability is a risk of both dementia 29,30 and cerebral SVD 31,32 .Thus, our findings suggest a multiplex linkage among PWV, EPVS, and cognitive impairment in DLB.Because we did not include participants with AD or Parkinson's disease in the present study, the mechanism of this multiplex linkage will need to be investigated in more detail in future studies.

CS-PVS
Another notable finding of the present study was that NfL was significantly associated with DLB, as has been previously reported 33 .However, CS-PVS was not as strongly associated with cognitive impairment in DLB as has been previously reported 7 .This discrepancy may be the result of the present study setting-in which we mainly enrolled participants with DLB as the dementia group-because CS-PVS is strongly associated with cerebral amyloid angiopathy 34 .In addition, we found that participants with BG-PVS were older and more likely to have hypertension, suggesting the presence of hypertensive cerebral SVD, as has been previously reported 34 .Lastly, the cut-off value of PWV for the detection of BG-PVS (17.5 m/s) was nearly the same as that reported for other subtypes of cerebral SVD, such as SLI (17.2 m/s) 19 , progressive acute lacunar stroke (18.2 m/s) 21 , and WMH (18.3 m/s) 20 .These findings are therefore also reasonable because these factors are all components of hypertensive cerebral SVD.The present study has several strengths.First, we revealed novel relationships among EPVS, PWV, and DLB.High PWV was strongly associated with EPVS, suggesting that high PWV increases cerebrovascular pulsatility, thus accelerating both hypertensive cerebral SVD and glymphatic dysfunction.Second, we revealed that BG-PVS was associated with DLB.These findings are novel because, although associations between DLB and various MRI findings (such as WMH or cerebral microbleeds [CMB]) have been discussed 8,35 , an association with EPVS has not been previously reported.Glymphatic dysfunction may also exist in DLB, and might accelerate α-synuclein aggregation.We offer new research directions regarding cerebral SVD (specifically, the glymphatic system and cerebrovascular pulsatility).Another strength is that we systematically evaluated cognitive function using a comprehensive geriatric assessment, several neuropsychological tests, and blood biomarkers.Our findings may thus shed light on the mechanisms of cerebrovascular pulsatility, and encourage further exploration of the relationships between cerebral SVD and dementia.
The present study also has several limitations.A causal relationship between PWV and EPVS was unable to be established because of the study's cross-sectional design.We were also unable to assess amyloid-β and α-synuclein in the current study because the Gerontological Investigation of Microbiome: a Longitudinal Estimation Study (the Gimlet study) 11 did not perform cerebrospinal fluid testing or positron emission tomography.4. Multivariable logistic regression analyses for the presence of DLB.The dependent variable was the presence of DLB.Note that BG-PVS ≥ 2 was defined as having a score ≥ 2 based on a magnetic resonance imaging scan at the level of the basal ganglia.Model 1: univariate analysis.Model 2: adjusted for age, sex, and pulse wave velocity (≥ 19 m/s).Model 3: adjusted for model 2 factors and components of cerebral small vessel disease (silent lacunar infarcts, white matter hyperintensity, and cerebral microbleeds).Model 4: backward stepwise multivariable logistic regression analysis adjusted for model 2 factors, education years, and risk factors (hypertension, dyslipidemia, diabetes mellitus, ischemic heart disease, a history of stroke, chronic kidney disease, smoking, alcohol consumption, and apolipoprotein E ε4).Model 5: backward stepwise multivariable logistic regression analysis adjusted for model 3 and 4 factors.to detect the presence of cognitive impairment.We also did not assess gray matter volume in the brain, which can be used to identify DLB subtypes 37 .Additionally, the combined inclusion of participants with DLB and those without dementia in our analyses of PWV and EPVS may have led to an overestimation of our interpretations.Moreover, the small number of participants with DLB and the large number of potential variables may have led to our study being statistically underpowered.Specifically, the small number of events per variable may have influenced the validity of the logistic model 38 .Selection bias may also exist because this was a single hospital-based cohort study in which participants agreeing to a fecal examination-the results of which were not used in the current study-were enrolled.
In conclusion, although this sub-study was a preliminary analysis, it provides evidence for relationships between EPVS, PWV, and DLB.BG-PVS was independently associated with DLB, and high PWV was associated with both BG-PVS and CS-PVS.High PWV may cause cerebrovascular pulsatility, which then accelerates EPVS and worsens cognitive impairment.Detailed assessments of the relationships between EPVS and PWV in DLB should be conducted in future studies to determine the underlying mechanisms.9) the ankle-brachial index as an indicator of arterial stiffness, and PWV as an indicator of arteriosclerosis 23 and the impact of pulse 25 ; and (9) nutritional and diet assessments 14 .Clinical samples and data were provided by the NCGG Biobank, which collects clinical data for research.

Brain imaging
Patients underwent 1.5 T MRI (Philips Ingenia, Eindhoven, Netherlands) of the brain.The obtained MRI scans included diffusion-weighted imaging, fluid-attenuated inversion recovery (FLAIR) imaging, T2-weighted imaging, T2 * -weighted gradient-echo imaging, three-dimensional T1-weighted sagittal and axial coronal views, and three-dimensional time-of-flight magnetic resonance angiography scans.VSRAD software (Eisai Co., Ltd., Tokyo, Japan) was used to quantify cortical and hippocampal atrophy; medial temporal structures involving the entire region of the entorhinal cortex, hippocampus, and amygdala show significant atrophy in patients with very mild AD, and can be specifically identified by a VSRAD software program 39 .Lower VSRAD values indicate less brain atrophy.One trained neurologist (N.S.) assessed all MR images and was blinded to participant data.

Cerebral SVD
The presence and components of cerebral SVD were categorized according to the Standards for Reporting Vascular Changes on Neuroimaging recommendations 4 .We defined an SLI as a focal lesion of ≥ 3 mm in diameter that was hyperintense on T2-weighted imaging and hypointense on FLAIR images.We defined WMH as an irregular periventricular hyperintensity (Fazekas grade ≥ 3) and/or early confluent or confluent separate deep hyperintense lesions (Fazekas grade ≥ 2) in the white matter on T2-weighted and FLAIR images.We defined a CMB as a focal area of signal loss in the brain parenchyma of < 5 mm on a T2*-weighted gradient-echo imaging scan.We defined EPVS as small (< 3 mm), punctate (if perpendicular to the plane of the scan) or linear (if longitudinal to the plane of the scan) hyperintensities on T2 images in the basal ganglia or centrum semiovale, as previously reported 34 .BG-PVS and CS-PVS were coded using the following scale, which was applied to standard axial images: 0 (no EPVS); 1 (< 10 EPVS); 2 (11-20 EPVS); 3 (21-40 EPVS); and 4 (> 40 EPVS) 40 .

Classification of cognitive function
Dementia was defined as an MMSE score < 20 and/or a CDR score ≥ 1, in accordance with the definitions used in our previous studies [10][11][12] .Participants who did not have dementia were further categorized as having either MCI or NC.MCI was defined as an MMSE score ≥ 20 and a CDR score of 0.5, which indicates possible, very mild dementia and a higher risk of developing dementia 10 .NC was defined as an MMSE score ≥ 20 and a CDR score of 0. Participants presenting with either MCI or NC were categorized into the non-dementia group in this sub-analysis.

Diagnosis with DLB
DLB was diagnosed according to criteria developed by the DLB Consortium 8 .To assess DLB-related motor and non-motor symptoms, UPDRS 41 scores and subscales were assessed by one trained neurologist (N.S.).

Measurement of NfL
Blood samples were collected and frozen at − 81 °C in the NCGG Biobank.NfL was assessed as a disease-nonspecific biomarker of neural damage.Plasma NfL concentrations were measured using the NF-Light Advantage

Statistical analysis
Continuous, ordinal, and categorical variables are expressed as means ± standard deviations, medians and interquartile ranges, and frequencies and proportions (percentages), respectively.These data were compared using Student's unpaired t-tests, Wilcoxon rank-sum tests, and χ 2 tests, respectively.We first compared clinical characteristics between women and men, and between participants with DLB and those without dementia.Second, we compared clinical characteristics between participants with EPVS and those without EPVS.In detail, we prespecified a dichotomized classification of BG-PVS as high (score ≥ 2) or low (score < 2) and of CS-PVS as high (score ≥ 3) or low (score < 3) according to the distributions of the participants in the present study and the categories used in previous studies 43,44 .Third, the relationships between BG-PVS, CS-PVS, and PWV were evaluated separately.Finally, univariate and multivariate logistic regression models were used to identify independent associations between: (1) DLB and EPVS (both BG-PVS and CS-PVS), and (2) EPVS and PWV.The ORs and 95% CIs were calculated.All comparisons were two-tailed, and p < 0.05 represented statistical significance.Data were analyzed using the JMP 18.0 software package (SAS Institute Inc., Cary, NC, USA).

Table 5 .
ORs of PWV in multivariable logistic regression analyses for the presence of BG-PVS.The dependent variable was the presence of BG-PVS (score ≥ 2 based on a magnetic resonance imaging scan at the level of the basal ganglia).Model 1: univariate analysis.Model 2: adjusted for age and sex.Model 3: backward stepwise multivariable logistic regression analysis adjusted for model 2 factors and risk factors (hypertension, dyslipidemia, diabetes mellitus, ischemic heart disease, a history of stroke, chronic kidney disease, smoking, and alcohol consumption).Model 4: backward stepwise multivariable logistic regression analysis adjusted for model 3 factors and components of cerebral small vessel disease (silent lacunar infarcts, white matter hyperintensity, and cerebral microbleeds).BG-PVS, enlarged perivascular spaces in the basal ganglia; CI, confidence interval; OR, odds ratio; PWV, pulse wave velocity.

Table 6 .
ORs of PWV in multivariable logistic regression analyses for the presence of CS-PVS.The dependent variable was the presence of CS-PVS (score ≥ 3 based on a magnetic resonance imaging scan at the level of the centrum semiovale).Model 1: univariate analysis.Model 2: adjusted for age and sex.Model 3: backward stepwise multivariable logistic regression analysis adjusted for model 2 factors and risk factors (hypertension, dyslipidemia, diabetes mellitus, ischemic heart disease, a history of stroke, chronic kidney disease, smoking, and alcohol consumption).Model 4: backward stepwise multivariable logistic regression analysis adjusted for model 3 factors and components of cerebral small vessel disease (silent lacunar infarcts, white matter hyperintensity, and cerebral microbleeds).CI, confidence interval; CS-PVS, enlarged perivascular spaces in the centrum semiovale; OR, odds ratio; PWV, pulse wave velocity.